This invention relates to a novel method for small sample preparation using a tube or column, such as a pipette tip, in which the interior surface is coated with a solid matrix for sample preparation. Said solid matrix is composed of a polymeric substance such as polytetrafluoroethylene (PTFE) and one or more column materials such as reactive or absorptive materials suited for sample filtration, separation or purification. The desired sample, containing bio-molecules such as DNA, proteins or other molecular components, is passed through said tube or column, which may be a pipette tip, or like structure.
Depending on the specifications of the column materials in the solid matrix, selected molecules from the sample can be separated or purified by binding to, or by being entrapped in, the column material components of the solid matrix. The bound molecules can later be eluted from the solid matrix by the use of different solvents. The tube described in the present invention has an opening at the top end, through which the sample is introduced into the tube and an open end at the bottom, through which selected components of the sample flow through during the sample separation process. Said tube may be of any shape or size in any configuration suitable for a given set of experimental conditions. The present invention is suited for samples with volumes from nanoliters to milliliters.
Although a spectrum of analytical methods for small sample separation and purification have been developed, a number of problems, such as the slow speed of the separation process and the loss of sample volumes, limit the quality of currently available methods. The present invention describes a small sample preparation method that both speeds up the sample purification and separation process and minimizes the extent of sample loss. This invention is a method for sample preparation that uses a tube or column where the interior surface of said tube or column is coated with a solid matrix. The solid matrix contains a polymeric substance such as polytetrafluoroethylene (PTFE), as well as, column materials such as reactive or absorptive materials suited for sample filtration, size-based separation or purification. The column material can be composed of chromatographic media such as gel-filtration, ion-exchange, reverse-phase, and silica or modified silica media.
As mentioned above, currently available methods for the separation and purification of micro volumes of samples often result in undesirable sample loss. Since the volumes of desired molecules, such as proteins or bio-molecules, are often very small, the loss of even small volumes in such samples can represent a significant portion of the total sample. In currently available methods, sample loss often results due to the presence of filters or other components in the separation column. For example, currently available methods that use a filter or chromatographic material plug at the bottom of a pipette tip often result in the loss of sample on the filter or in the matrix containing the chromatography material. Since the volume of such a filter or plug may sometimes be as large as the volume of the micro sample itself, sample loss can be quite significant and is often accompanied by a slowed rate of separation. Also, different solvents interact differently with the filter itself further adding variation to the quality of the separation or purification of a particular sample.
One method that is currently available is the ZipTip developed by Millipore. This system consists of a micropipette tip that contains a cast of the column material in a porous matrix that is formed as a plug at the lower open end of the tip. Since the casted material plugs the open end through which the sample is pulled into the tip, however, the flow of the sample through the plug and into the tip may be slowed down or impeded by the plug. Furthermore, when this system is used in a multi-sample configuration such as a 96-well plate, there may be inconsistency in the quantity of sample that is absorbed into the different tips on the same plate and in the quality of the sample separation process itself.
In the invention described herein, the solid matrix is applied to the tube or pipette tip such that it coats the interior sides of the tube without significantly obstructing the flow of the sample through the lower opening of the tip. The solid matrix may be affixed to the interior walls of the tube using any physical or chemical methods that include, but are not limited to adhesion, heat, pressure and etching. For optimal sample separation, the sample can be aspirated back and forth multiple times to ensure optimal binding of the desired bio-molecules to the column material in the solid matrix. The bio-molecules can then be eluted from the solid matrix using different solvents.
The solid matrix coating is composed of one or more inert materials such as PTFE (polytetrafluoroethylene) and the desired column materials. Said desired column materials adhere to the inner surface of the tube when used in combination with said inert materials resulting in a solid matrix that is effective for sample separation. Sample separation and purification tubes designed with such an interior coat of the solid matrix are highly effective because the sample can flow more easily through the tube, column or pipette tip chamber and it is in contact with a greater surface area of the coated solid matrix containing the column material. The quality of sample preparation is also enhanced due to increased consistency in performing the same procedure whether in a single or simultaneous, multi-tip framework.
The various features of novelty, which characterize the invention, are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its advantages and objects, reference is made to the accompanying drawings and descriptive matter in which a preferred embodiment of the invention is illustrated.